Genetic Burden of TNNI3K in Diagnostic Testing of Patients With Dilated Cardiomyopathy and Supraventricular Arrhythmias

BACKGROUND: Genetic variants in TNNI3K (troponin-I interacting kinase) have previously been associated with dilated cardiomyopathy (DCM), cardiac conduction disease, and supraventricular tachycardias. However, the link between TNNI3K variants and these cardiac phenotypes shows a lack of consensus concerning phenotype and protein function. METHODS: We describe a systematic retrospective study of a cohort of patients undergoing genetic testing for cardiac arrhythmias and cardiomyopathy including TNNI3K. We further performed burden testing of TNNI3K in the UK Biobank. For 2 novel TNNI3K variants, we tested cosegregation. TNNI3K kinase function was estimated by TNNI3K autophosphorylation assays. RESULTS: We demonstrate enrichment of rare coding TNNI3K variants in DCM patients in the Amsterdam cohort. In the UK Biobank, we observed an association between TNNI3K missense (but not loss-of-function) variants and DCM and atrial fibrillation. Furthermore, we demonstrate genetic segregation for 2 rare variants, TNNI3K-p.Ile512Thr and TNNI3K-p.His592Tyr, with phenotypes consisting of DCM, cardiac conduction disease, and supraventricular tachycardia, together with increased autophosphorylation. In contrast, TNNI3K-p.Arg556_Asn590del, a likely benign variant, demonstrated depleted autophosphorylation. CONCLUSIONS: Our findings demonstrate an increased burden of rare coding TNNI3K variants in cardiac patients with DCM. Furthermore, we present 2 novel likely pathogenic TNNI3K variants with increased autophosphorylation, suggesting that enhanced autophosphorylation is likely to drive pathogenicity.


RNA (splice) analysis
Total RNA was obtained from peripheral blood using PAXgene tubes and the PAXgene blood RNA kit (Qiagen). cDNA was produced by reverse transcription-PCR with the use of Superscript III kit (Invitrogen) and random hexamers. PCRs were performed using TNNI3K

Enrichment analysis Amsterdam UMC
For the enrichment analysis in the Amsterdam UMC panels, a chi-square test was performed. The threshold for significance was defined as p<0.008 (=0.05/6 disease endpoints) corrected for multiple testing with the Bonferroni method.

TNNI3K disease associations in the UK Biobank
For each phenotype, we used the Cauchy distribution test 37 to combine the various pvalues into a single p-value for the analysis (Supplemental Figure I). The Cauchy distribution test allows for valid aggregation of multiple, potentially correlated, test statistics into a single omnibus test statistic 37 . The Bonferroni-corrected significance cutoff for the overall analysis was set at 0.00714 (α=0.05/7 disease endpoints). Finally, significant results were assessed in a sensitivity analysis restricted to individuals of European ancestry only.

Auto-phosphorylation assay
Data are expressed as mean ± standard error of the mean (SEM). Comparison of autophosphorylation levels that followed normal distributed was performed with one-way repeated highlighted in blue) and 5 tests for LoF variants combined with each of the missense masks (highlighted in purple). Then, p-values for each of the LoF tests were combined into a single p-value using the Cauchy distribution test, as were each of the missense tests and each of the LoF+missense tests. Prior to combining p-values, tests with cMAC<20 were removed. Finally, to produce a single p-value for a given phenotype, we combined the LoF, missense and LoF+missense p-values into a single p-value, again using the Cauchy distribution test.

TNNI3K-p.Ile512Thr family
The proband (IV-4) is a male carrier of TNNI3K-p.Ile512Thr experienced syncope during exercise at the age of 22. His medical history includes recurrent syncopes while ill. His ECG revealed RBBB and LAD. Echocardiographic examination did not reveal any abnormalities. The ajmaline test was negative. His brother (IV-5) also carried the variant and presented with iRBBB and LAD with no history of syncope or any abnormal echocardiographic findings. Their mother (III-3), carrier of the variant, suffered from syncopes since the age of 14. Her ECG shows LBBB and LAD. Her echocardiographic examination showed borderline systolic function (LVEF=51%) with normal dimensions of the ventricles, first to second-grade tricuspid insufficiency and mild dilatation of the right atrium. Her mother (II-3) was diagnosed with LBBB at the age of 46 and later developed DCM (LVEF=30%) followed by cardiac resynchronization therapy defibrillator (CRT-D) implantation. She also tested positive for the variant. Family history was also positive for multiple cases of AF and SCD at a young age.

Family 1
The proband is a 69-year-old man (1.II-4), who carries TNNI3K-p.His592Tyr. At the age of 51, the patient was admitted to the Cardiology Department with dyspnea and diagnosed with DCM, severe mitral insufficiency (MI), and a left ventricular ejection fraction (LVEF) of 20%. His first 12-lead ECG at admission revealed 1 st grade AV block (PQ-interval=220ms), LBBB, LAD, and abnormal ST-segments. He underwent a pacemaker implantation two years later and his LVEF increased over time to 35% with an improvement of mitral valve function (MI from 3rd to 2nd grade). Further genetic screening of this family detected the TNNI3Kp.His592Tyr in both daughters of the proband. Anamnestic indication of further cardiomyopathy in the family had no follow-up due to lack of informed consent.

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The 48-year-old daughter (1.III-2), who was found to be a carrier of the variant, presented with mild LV dysfunction with round morphology of LV at the age of 32. Her echocardiographic examination at admission showed normal dimensions and decreased function of the LV. Magnetic resonance imaging (MRI) of the heart revealed decreased LVEF (50-63%). Over the years, heart function and morphology did not worsen. Her latest echocardiographic examination showed round LV morphology with LVEF=45%. Her treatment included candesartan. She suffers from palpitations and fatigability.
The son of the proband (1.III-3) demonstrated no symptoms at age of 46. His echocardiographic examination showed LVEF=46% with normal dimensions of all cardiac chambers, normal diastolic function, and LVEDD=49mm. His ECG and Holter examination did not reveal any conduction abnormalities or arrhythmias. He is not a carrier of the variant.
The younger daughter of the proband (1.III-4) presented with mild DCM with borderline dimensions, round morphology of LV, and decreased function of LV at the age of 33. MRI confirmed these findings (LVEF=46% and LVEDD=55mm), showing no signs of fibrosis. 12lead ECG, Holter examination, and a stress test did not detect any abnormalities. She also carries the TNNI3K-p.His592Tyr variant.

Family 2
The 68-year-old proband (2.II-1) experienced out-of-hospital cardiac arrest (OHCA) and was diagnosed with DCM at the age of 54. Echocardiography and MRI showed dyssynchrony and reduced LVEF equal to 25% and 43%, respectively. Her treatment included an implantable cardioverter-defibrillator (ICD) implantation. ECG recordings revealed LBBB and borderline first-degree AV block (PR=200ms).
Her mother (2.I-3) was diagnosed with a third-grade AV block at the age of 65, which was followed by pacemaker implantation. The TNNI3K variant His592Tyr was also detected in her.

Family 3
The proband is a 33-year-old woman (3.II-1) with a history of broad complex tachycardia (most probably AVNRT with aberrant conduction) during the exercise test at the age of 27. A year later she developed atrial flutter and atypical AVNRT, for which she underwent ablation. Her exercise test showed broad complex tachycardia with LBBB morphology (suspected non-sustained ventricular tachycardia (nVT)) with no abnormalities in MRI and echocardiographic examination. After re-ablation her ECG revealed a ventricular escape rhythm. Holter examination revealed frequent symptomatic PVCs (palpitations during PVCs). Her medical history also included AV block and well-documented vasovagal syncope.
Her latest examinations did not reveal any arrhythmias or morphological abnormalities. She also experienced syncope, which was evaluated to be of vasovagal origin. Her mother (3.I-2) tested negative for the TNNI3K variant and did not demonstrate any cardiac arrhythmias or cardiomyopathy. She did have an aortic valve insufficiency.

Family 4
The proband is a 52-year-old woman (4.II-2) with a diagnosis of peripartum cardiomyopathy with hemodynamic decompensation (LVEF=28%) at the age of 34. LV function recovered partially with dilatation of the LV and LA and sinus tachycardia at rest.
Seven years later she demonstrated recurrent AVNRT at exercise. At the age of 47, she underwent an unsuccessful ablation of a slow pathway, followed by possible junctional tachycardia. Her Holter examination was associated palpitations with episodes of junctional tachycardia. A year later the ablation procedure was repeated and the patient did not present any symptoms of SVTs. The 12-lead ECG did not reveal any abnormalities within 3 months after ablation. Additionally to the TNNI3K variant, she is a carrier of a variant in RBM20c.3115C>T p.(Pro1039Ser), which is of unknown significance. The first daughter of the proband (4.III-I), tested positive for the TNNI3K variant, her clinical examination did not reveal any abnormalities. Her younger sister tested negative for the variant and is healthy (4.III-2).

Family 5
The proband is a 68-year-old woman (5.II-2) with a long history of DCM and recurrent arrhythmias. Since the age of 58, she experienced syncope during episodes of atrial tachycardias, for which she underwent a partially successful ablation. Her echocardiographic examination revealed mild diastolic dysfunction. Six years later she started experiencing broad complex tachycardia episodes (Sustained VT with LBBB aberrant conduction). Her history also includes paroxysmal AF with spontaneous conversion and frequent PVCs. After the second catheter ablation, she did not suffer from palpitations. The latest MRI showed LV dilation, LVEF=34%, and LVEED=62mm. The latest echocardiographic examination suggested a mild concentric remodeling with normal dimensions of the heart chambers. One of the daughters (5.III-2) was shown to carry the variant. Cardiac examination (at age 41) did not demonstrate any cardiac arrhythmias or cardiomyopathy.

Family 6
The proband (6.V-1) carries the TNNI3K-p.His592Tyr variant and died suddenly at the age of 56. He was diagnosed with DCM and AF at the age of 52. His brother (6.V-2) also carries the variant and showed VT during an exercise test and underwent ICD implantation. His sister (6.V-3) has a history of AF episodes, her genetic status is unknown. Their mother (6.IV-2) is also a carrier of the variant in TNNI3K and experienced cardiac arrhythmias (palpitations) at the age of 43. Later, she developed paroxysmal AF, nVTs, and LBBB. She also underwent a ICD implantation. Her mother (6.III-1) was diagnosed with arrhythmias at age 37 and died from SCD at age 41. The family history further includes multiple cases of SCD at a young age (see figure 3F).